We are interested in the cell biology of cells that make steroid hormones, and hope to follow labeled steroid substrates and products through these cells by autoradiography at the light and electron microscope levels, and thus show how these materials move within the cells during synthesis and secretion. By this means, we hope to gain information about the time course of these processes, the location of the enzymes (by using specific inhibitors), substrate pools, and perhaps sites of regulation. Since steroids and their precursors are diffusible substances, routine autoradiography is not feasible--the substances would be removed or at least displaced by the technique. Over the last few years the principal investigator has developed a method for cutting frozen thin sections of fresh-frozen tissue for electron microscopy. These sections are prepared under conditions that should maintain diffusible substances in place. A method for carrying out the EM autoradiography on these sections has been developed. The accumulation of testosterone metabolites in liver canaliculi is being used as a test system to see how well diffusible substances are maintained in place during autoradiography. Further biochemical work is necessary to establish which steroid-secreting organs in various species are most effective in converting introduced substrates to steroid hormones, for the eventual completion of the project described in the first paragraph. In a second major project, we are attempting to localize steroid receptor proteins by conventional autoradiography at the light and possibly the electron microscope levels. Dialdehyde fixatives (e.g., glutaraldehyde) may possibly stabilize steroid on the receptor to some degree. Androgen receptors in rat ventral prostate and seminiferous tubules will be studied with the method.